FileNotFoundError

[cculma]

Hi, I was running SQANTI3 but it shows me this message: No such file or directory: ### FileNotFoundError: [Errno 2] No such file or directory: '/home/hawkins/Documents/Cesar/RNA/globus/lordec_reports/sqanti3/refAnnotation_sqanti_LID50568_1001.genePred'

Do you know what could be wrong?

Best,

Cesar

[FJPardoPalacios]

Hi Cesar,

Could you give me more information about how did you run SQANTI3?
It seems to be a problem with some path, can you check them?

Fran

[cculma]

Hi, I am running the bed file resulting from TAMA collapse as follow:

python ../../../Iso_assay/tama/tama/tama_go/format_converter/tama_convert_bed_gtf_ensembl_orf_nmd.py no_cap_Ms_LID50568_1001.bed no_cap_Ms_LID50568_1001.gtf

And then I am using the gtf file as input to run SQANTI3:

python ../../../Iso_assay/sqanti2/SQANTI3/sqanti3_qc.py \
-x /home/hawkins/Documents/Cesar/NGSEP/ngsep_tutorial/Ms_Chen/Ms/Ms \
-e /home/hawkins/Documents/Cesar/RNA/globus/lordec_reports/rna_seq/kallisto_LID50568_1001/abundance.tsv \
-c /home/hawkins/Documents/Cesar/RNA/globus/lordec_reports/star_Ms/SJ.out_LID50568_1001.tab \
-d /home/hawkins/Documents/Cesar/RNA/globus/lordec_reports/sqanti3 \
-t 12 \
-o sqanti_LID50568_1001 \
-g no_cap_Ms_LID50568_1001.gtf \
/home/hawkins/Documents/Cesar/NGSEP/ngsep_tutorial/Ms_Chen/final.all.gene.gff \
/home/hawkins/Documents/Cesar/NGSEP/ngsep_tutorial/Ms_Chen/final.all.fasta 

I followed all the tutorial installation, step by step, and additionally, if I used the file resulting from collapse with cupcake, cupcake.gff it works fine with sqanti1.

Best,

Cesar

[veravelasco]

Hi, I was running SQANTI3 but it shows me this message: No such file or directory: ### FileNotFoundError: [Errno 2] No such file or directory: '/home/hawkins/Documents/Cesar/RNA/globus/lordec_reports/sqanti3/refAnnotation_sqanti_LID50568_1001.genePred'

Do you know what could be wrong?

Best,

Cesar

Hi. I have the same error now. How'd you fix it? Thanks.

Vera

[cculma]

In my case were two errors:

1. The wrong use of genome.gff instead of genome.gtf
2. I was trying to run SQANTI3 in the workstation with python2.7 by default (it works well for SANTI1), therefore I install SQANTI3 in the server which runs with python3.7 and now is working.

Best,

Cesar

[Magdoll]

Closed unless otherwise noted by OP.

[Ismail-Curie]

Hello, I still have the same issue, could you help me find a solution to that.

[Magdoll]

Hi @Ismail-Curie , please show the full command you are running and the error. Also, please make sure you are using the latest SQANTI3 version (v1.1)

[Ismail-Curie]

Hi @Magdoll,

I'm using the latest SQANTI3 version, below the full command:
python3.7 sqanti3_qc.py --force_id_ignore /home/ismail/Downloads/cluster_via_merge/clustered.lq.fasta /home/ismail/Downloads/ref/gencode.v28.annotation.gtf /home/ismail/Downloads/ref/hg38_base.fasta -x /home/ismail/my_hg38_base/my_hg38_base -t 10 -d /home/ismail/ALTERNA/Results_SQANTI/

Thank you,
Ismail

[Magdoll]

Hi @Ismail-Curie ,
I'm actively moving users away from inputting a fasta input - can you please provide a GTF input instead? You can align clustered.lq.fasta using minimap2 and convert it to GTF/GFF3 using sam_to_gff3.py in Cupcake.

If issue persists, please provide an email so I can request file upload to check for issues :-)

-Liz

[Ismail-Curie]

Hi @Magdoll,
I still have the same error even after using a gtf input instead of fasta, can I send you the files? (my email is jamail.ismail1@gmail.com)
Thank you,
Ismail

[Magdoll]

Hi @Ismail-Curie , file request emailed!

[Ismail-Curie]

Hi @Magdoll ;
I sent the input file.
Thank you.

[Magdoll]

Hi @Ismail-Curie ,
I discovered two issues and both are related to your GFF3 input structure.

The input needs to have a transcript record and a exon record. I converted it using gffread

gffread -T input.gff3 -O output.gtf

Next, some of your transcripts have the same ID and are on different chromosomes or strand.
For example below you have three records that share the same ID, this causes errors in running SQANTI3.

I am emailing you back the fixed results.

chr1    hg38    transcript      634766  634924  .       +       .       transcript_id "transcript/455494"; gene_id "transcript/455494";
chr1    hg38    exon    634766  634924  .       +       .       transcript_id "transcript/455494"; gene_id "transcript/455494";
chrM    hg38    transcript      9546    9990    .       +       .       transcript_id "transcript/455494"; gene_id "transcript/455494";
chrM    hg38    exon    9546    9990    .       +       .       transcript_id "transcript/455494"; gene_id "transcript/455494";
chrM    hg38    transcript      9571    9787    .       -       .       transcript_id "transcript/455494"; gene_id "transcript/455494";
chrM    hg38    exon    9571    9787    .       -       .       transcript_id "transcript/455494"; gene_id "transcript/455494";

[Ismail-Curie]

Thank you very much @Magdoll.

[skudashev]

Hello @Magdoll, I am trying to solve the same issue and have converted my collapsed.gff from cupcake to a .gtf, but am still getting the same error.

**** Running SQANTI3...
**** Parsing provided files....
Reading genome fasta /ei/projects/3/31655266-640a-41d2-8663-59bba38bc3c4/data/data/References/hg38_sequin.fa....
Skipping aligning of sequences because GTF file was provided.

Indels will be not calculated since you ran SQANTI3 without alignment step (SQANTI3 with gtf format as transcriptome input).
**** Predicting ORF sequences...
**** Parsing Reference Transcriptome....
Traceback (most recent call last):
  File "/ei/.project-scratch/8/8289c66d-2d56-4706-a307-5a9a3eb3747e/SQANTI3/sqanti3_qc.py", line 2461, in <module>
    main()
  File "/ei/.project-scratch/8/8289c66d-2d56-4706-a307-5a9a3eb3747e/SQANTI3/sqanti3_qc.py", line 2444, in main
    run(args)
  File "/ei/.project-scratch/8/8289c66d-2d56-4706-a307-5a9a3eb3747e/SQANTI3/sqanti3_qc.py", line 1839, in run
    refs_1exon_by_chr, refs_exons_by_chr, junctions_by_chr, junctions_by_gene, start_ends_by_gene = reference_parser(args, list(genome_dict.keys()))
  File "/ei/.project-scratch/8/8289c66d-2d56-4706-a307-5a9a3eb3747e/SQANTI3/sqanti3_qc.py", line 664, in reference_parser
    for r in genePredReader(referenceFiles):
  File "/ei/.project-scratch/8/8289c66d-2d56-4706-a307-5a9a3eb3747e/SQANTI3/sqanti3_qc.py", line 137, in __init__
    self.f = open(filename)
FileNotFoundError: [Errno 2] No such file or directory: '/ei/.project-scratch/8/8289c66d-2d56-4706-a307-5a9a3eb3747e/refAnnotation_Capture4_barcode11_sqanti.genePred'

I ran cDNACupcake with the following command:
python cDNA_Cupcake/cupcake/tofu/collapse_isoforms_by_sam.py --input Capture4/barcodes/demultiplexed/barcode11/barcodes11.fastq --fq -s Capture4_barcodes11.sorted.sam --dun-merge-5-shorter -c 0.97 -o Capture4_barcode11
And then SQANTI:
python SQANTI3/sqanti3_qc.py Capture4_barcode11.collapsed.gtf References/gencode.v29.annotation_sequin.gtf References/hg38_sequin.fa --cpus 20 --report pdf --genename --isoAnnotLite -o Capture4_barcode11_sqanti

I also checked my collapsed.gtf and there don't seem to be any duplications of same IDs.

[Ismail-Curie]

Hello @kudasonya,
The problem might be related to your fasta file, I had the same issue for a long time, it was resolved by changing my reference.
good luck,

(or the annotation file, I don't remember exactly)
Ismail